This Bioengineering Research Group R01 competing continuation proposal aims to continue the productivity and impact of the preceding funded period in elucidating key surface properties of commercial and model nucleic acid microarray diagnostic assays. Despite immense potential clinical and environmental applications for these multiplexed diagnostic formats, only two microarray assays are FDA-approved for clinical diagnostic use. Array surface-capture problems preclude assay reliability, sensitivity, and reproducibility, occurring across all commercial platforms, within batches in a single platform, between different clinical labs, and even within microarray elements on a single slide. This is also the most significant problem for a substantial fraction of assays under commercial development: custom printed arrays involving different printing technologies and assay reagents. Hence, clinical impact of microarrays is currently very limiting. Significance is derived from this proposal's focus on obtaining new information necessary for understanding and resolving the issues of assay reliability, sensitivity and reproducibility. Wide adoption of microarray technology for recent FDA, EPA and NIH Critical Path initiatives for disease, drug pharmacology and toxicitiy screening goals relies on improving assay performance and developing clear understanding of their limitations. This proposal responds directly to that need. The working hypothesis is that precise quantitation of surface immobilization and capture efficiencies in microarrays comprising nucleic acids using newly available high-resolution instrumentation will identify current assay limitations, and facilitate improved surface-capture assay performance necessary for clinical use of these diagnostics. Specific aims for the 3-year project include: Aim 1. Validate and correlate new surface analytical technology methods for molecular analysis of nucleic acids, specifically, to quantify printed and captured nucleic acid density on microarrayed surfaces. Aim 2. Compare DNA and PNA microarray printed spot uniformity as it relates to printed substrate conditions and chemistry, and address spot-to-spot variability at high chemical resolution as common sources of microarray assay variance and uncertainty observed on commercial microarray substrates. Aim 3. Improve standard microarray assay performance requirements and capture assay detection limits by direct quantitation and detection of small amounts of target DNA in complex media without pre-purification. This proposal focuses on improving the current capabilities in nucleic acid microarray bioassay. A suite of complementary state-of-the-art surface analytical methods will be exploited to assess variables affecting DNA surface immobilization and target hybridization in model and commercial assay formats. The strategy proposed addresses important bio-analytical problems in these assays to identify improvements that impact high-priority science and technology involved in monitoring public health and disease, pharmacology and personalized medicine, and toxicology. Developing reliable bio- assays capable of detecting DNA hybridization from real-world samples at an attomole level would allow rapid clinical diagnostic applications beyond current research use. Facilitating translation of these widely used microarray assay formats from current research to true clinical future use is a major aim of this research.